1. Field of the Invention
The present invention is related to primers for diagnosis of one or more kinds of cancers expressing the MAGE or GAGE genes or their subtypes, and a diagnostic kit containing the above primers. In a particular embodiment, the invention relates to common primers that can simultaneously detect six MAGE sub-types from MAGE 1 to MAGE 6 (MAGE 1–6) or eight GAGE sub-types from GAGE 1 to GAGE 8 (GAGE 1–8), a diagnostic kit containing the above common primers, and methods for use of these primers to detect cancer. Additionally, the invention includes methods for use of primers for detection of MAGE or GAGE expression in a clinical methodology to detect the expression of multiple MAGE and GAGE genes in biological samples, including patient samples for screening, diagnosis, or prognosis in cancer patients. The methodology may also include qualitative correlation of expression of the MAGE or GAGE genes to disease or quantitative measurement of expression of one or more genes to standard or control values, and the use of this comparison in the methodologies listed above.
2. Description of the Prior Art
The diagnosis of cancers has been accomplished through the medical physical examination, X-ray and CT, histological examination, etc. However, these methods have not been appropriate for detection of cancers at the initial stage, for discrimination between a malignant cancer and a benign tumor. It is believed that the initial stages of cancer development cannot be detected by direct physical observation, but occur at the genetic level where a number of genes that are involved in the onset or progression of the cancer are differentially expressed in the very earliest stages of the disease. In some cases, the initial stages of cancer may be marked by the expression of specific genes that either accompany the onset of the disease, or that are the actual causative agent in the onset or progression of cancer. Unfortunately, the ability to detect these events is complicated by the fact that hundreds or thousands of genes may be expressed during the onset of the disease and it is extremely difficult to detect and to separate normal gene expression from the expression of the specific genes that indicate or are responsible for, the onset or progression of disease.
However, recently developed molecular biological diagnostic methods have contributed greatly to the development of cancer diagnostics that are both specific and highly sensitive. The most widely used method among many molecular biological diagnostic methods is the polymerase chain reaction (PCR) or the reverse transcriptase-polymerase chain reaction (RT-PCR). In these techniques, abnormal genes, such as cancerous antigen genes expressed in a sample, are amplified and detected.
Particularly, RT-PCR is a method of detecting mRNA which is expressed by a particular gene. This method may be used for the diagnosis of a cancer by examining the property of expression of cancerous antigen genes. The most important matter in the diagnosis of a cancer in the RT-PCR method is the selection of the target gene for detection (hereinafter referred to as a “cancer diagnosis marker”). Usually, only one cancer diagnosis marker is selected and detected by this method.
The requirements for a cancer diagnosis marker are that it should be selectively expressed in cancerous tissue and should be expressed in large amounts in as many cancer types as possible. MAGE (melanoma antigen gene) and GAGE (‘G’ antigen gene) are two kinds of cancer-associated testis antigens. Selected subtypes or subfamilies of these genes are expressed in many kinds of cancer tissues, but are not expressed in most normal tissues, except for the testis. It has been clarified that MAGE has been expressed in many cancers such as the stomach cancer (1, 2), esophagus cancer (3), colon cancer (4), lung cancer (5), breast cancer (6, 7), liver cancer (8), leukemia (9), neuroblastoma (10), ovary cancer (11), etc. in many studies since it was discovered in the melanoma. And it has been reported that GAGE has been expressed in the melanoma, sarcoma, small-cell carcinoma, head and neck cancer, bladder cancer, ovary cancer, etc. (11, 12). MAGE or GAGE genes may be identified with distinct nomenclature as subtypes or subfamilies, such as MAGE A–L, and specifically such as MAGE A1–A15, MAGE B1–17, MAGE C1–7, MAGE D1–4, MAGE E1–3, MAGE F, MAGE G, MAGE H, MAGE 11–12, MAGE J1, MAGE K1, MAGE L2 and NECDIN.
Therefore, selected MAGE and GAGE genes are very extensively utilized as cancer diagnostic markers because detection of MAGE and/or GAGE expression detects many kinds of cancers and has a high specificity for expression in cancer tissues compared to other cancer-associated antigens such as the carcinoembryonic antigen, etc. Also, MAGE and GAGE have a high homogeneity of gene sequence among gene subtypes. About 12 kinds of sub-types of MAGE have the homogeneity ranging from about 56% to 99%, and 8 kinds of sub-types of GAGE have the homogeneity ranging from about 82% to 99%. Therefore, it is possible to increase the cancer diagnosis rate by using such same DNA sequence part as a primer since many sub-types of MAGE or GAGE may be detected simultaneously during RT-PCR. De Plaen et al., “Structure, chromosomal localization, and expression of 12 genes of the MAGE family, Immunogenetics 1994;405(5):360–9; Chomez, Patrick et al., “An Overview of the MAGE Gene Family with the Identification of all Human Members of the Family,” Cancer Research 61, 5544–5551, Jul. 15, 2001; Barker, P A, et al., “The MAGE proteins: emerging roles in cell cycle progression, apoptosis, and neurogenetic disease,” J. Neurosci Res 2002 March 15:67(6):705–12; Van den Eynde, B., et al., “A new family of genes coding for an antigen recognized by autologous cytolytic T lymphocytes on a human melanoma,” J. Exp. Med. 1995 September 1,;182(3):689–98; and De Backer, Olivier, “Characterization of the GAGE Genes That Are Expressed in Various Human Cancers and in Normal Testis,” Cancer Research 59, 3157–3165, Jul. 1, 1999).
Accordingly, the highly conserved, high homogeneity of sequences among MAGE and GAGE subtypes allows the design of common primers of the invention having a high selectivity and specificity for cancer detection and diagnosis. Moreover, the primers of the invention exhibit high specificity for cancer detection and diagnosis through simultaneous detection of many MAGE or GAGE gene subtypes.